This invention relates to a method of delivery of antisense oligonucleotide to a preselected locus in vivo, useful in the treatment of disease.
In the last several years, it has been demonstrated that oligonucleotides are capable of inhibiting the replication of certain viruses in tissue culture systems. For example, Zamecnik and Stephenson, Proc. Natl. Acad. Sci. U.S.A.,75:280-284 (1978), showed oligonucleotide-mediated inhibition of virus replication in tissue culture, using Rous Sarcoma Virus. Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A.,83:4145-4146 (1986), demonstrated inhibition in tissue culture of the HTLV-III virus (now HIV-1) which is the etiological agent of AIDS. Oligonucleotides also have been used to suppress expression of selected non-viral genes by blocking translation of the protein encoded by the genes. Goodchild, et al., Arch. Biochem. Biophys.,264:401-409 (1988) report that rabbit-globin synthesis can be inhibited by oligonucleotides in a cell-free system. Treatment with antisense c-myb has been shown to block proliferation of human myeloid leukemic cell lines in vitro. G. Anfossi, et al., Proc. Natl. Acad. Sci. USA, 86:3379 (1989).
A drawback to this method is that oligonucleotides are subject to being degraded or inactivated by cellular endogenous nucleases. To counter this problem, some researchers have used modified oligonucleotides, e.g., having altered internucleotide linkages, in which the naturally occurring phosphodiester linkages have been replaced with another linkage. For example, Agrawal et al., Proc Natl. Acad. Sci. U.S.A.,85:7079-7083 (1988) showed increased inhibition in tissue culture of HIV-1 using oligonucleotide phosphoramidates and phosphorothioates. Sarin et al., Proc. Natl. Acad, Sci. U.S.A.,85:7448-7451 (1988) demonstrated increased inhibition of HIV-1 using oligonucleotide methylphosphonates. Agrawal et al., Proc. Natl. Acad. Sci. U.S.A.,86:7790-7794 (1989) showed inhibition of HIV-1 replication in both early-infected and chronically infected cell cultures, using nucleotide sequence-specific oligonucleotide phosphorothioates. Leither et al., Proc. Natl. Acad. Sci. U.S.A.,87:3430-3434 (1990) report inhibition in tissue culture of influenza virus replication by oligonucleotide phosphorothioates.
Oligonucleotides having artificial linkages have been shown to be resistant to degradation in vivo. For example, Shaw et al., in Nucleic Acids Res.,19:747-750 (1991), report that otherwise unmodified oligonucleotides become more resistant to nucleases in vivo when they are blocked at the 3' end by certain capping structures and that uncapped oligonucleotide phosphorothioates are not degraded in vivo.
While antisense oligonucleotides have been shown to be capable of interfering selectively with protein synthesis, and significant progress has been made on improving their intracellular stability, the problem remains that oligonucleotides must reach their intended intracellular site of action in the body in order to be effective. Where the intended therapeutic effect is a systemic one, oligonucleotides may be administered systemically. However, when it is necessary or desirable to administer the oligonucleotide to a specific region within the body, systemic administration typically will be unsatisfactory. This is especially true when the target mRNA is present in normal cells as well as in the target tissue, and when antisense mRNA binding in normal cells induces unwanted physiological effects. Stated differently, the dosage of antisense oligonucleotide administered systemically that is sufficient to have the desired effect locally may be toxic to the patient.
An example of a treatment strategy which could greatly benefit from development of a method of limiting the effect of antisense oligonucleotide to a target tissue is the inhibition of smooth muscle cell proliferation which leads to restenosis following vascular trauma.
Smooth muscle cell proliferation is a poorly understood process that plays a major role in a number of pathological states including atherosclerosis and hypertension. It is the leading cause of long-term failure of coronary and peripheral angioplasty as well as of coronary bypass grafts.
Vascular smooth muscle cells in adult animals display a well defined phenotype characterized by an abundance of contractile proteins, primarily smooth muscle actin and myosins, as reviewed by S. M. Schwartz, G. R. Campbell, J. H. Campbell, Circ. Res., 58,427 (1986), and a distinct lack of rough endoplasmic reticulum. When subjected to injury in vivo or placed in an in vitro cell culture, adult smooth muscle cells (SMC) undergo a distinct phenotypic change and lose their "differentiated" state. The cells acquire large amounts of endoplasmic reticulum and gain actively synthesizing extracellular matrix. In addition, they begin expressing a number of new proteins including non-muscle myosins and actins, and PDGF A chain, as reported by R. J. Dilley, et al., Atherosclerosis, 63:99 (1987), P. Libby, et al., N. Engl. J. Med., 318:1493 (1988), while the expression of smooth muscle-specific contractile proteins such as smooth muscle myosin heavy chain and alpha actin decline, as shown by M. Kuro-o, et al. J. Biol. Chem., 264:18272 (1989) and A. W. Clowes, et al. J. Cell. Biol., 107:1939 (1988).
A nuclear oncogene c-myb may play an important role in these changes. The oncogene is homologous to the transforming gene of the avian myeloblastosis virus. Although considered originally to be expressed only in hematopoietic cells, c-myb has been shown to be present in chick embryo fibroblasts as well as in proliferating SMCs. C. B. Thompson, et al. Nature, 319:374 (1986); C. F. Reilly, et al. J. Biol Chem., 264:6990 (1989). The human c-myb gene has been isolated, cloned and sequenced. Majello et al., Proc. Natl. Acad. Sci. USA, 83:9636-9640 (1986). The expression of c-myb is growth-dependent. It is present in a low level in quiescent cells but increases rapidly as cells begin to proliferate and peaks near the late G.sub.1 phase of the cell cycle. C. F. Reilly, et al. J. Biol. Chem., 264:6990 (1989). Furthermore, expression of c-myb appears to correlate with the differentiation state of the cell. Myeloid erythroleukemia cells have been induced to differentiate and thereby decrease c-myb expression.
Heparin, as well as closely related heparin proteoglycans, can block smooth muscle cell proliferation in vivo as well as in vitro. A. W. Clowes, M. J. Karnovsky Nature, 265:625 (1977); C. R. Reilly, et al., J. Cell Physiol., 129, 11 (1986); J. R. Guyton, et al., Circ. Res., 46, 625 (1980); and L. M. S. Fritze, et al., J. Cell. Biol., 100:1041 (1985). This block occurs in a late G.sub.1 phase of the cell cycle and is associated with a decrease in the level of c-myb (but not that of c-fos or c-myc) expression, (C. F. Reilly, et al., J. Biol. Chem., 264:6990 (1989)), and a partial return of expression of smooth muscle specific contractile proteins. M. Kuro-o, et al., J. Biol. Chem., 264:18272 (1989) and A. W. Clowes, et al. J. Cell. Biol., 107:1939 (1988). Since c-myb appears to be critically involved in the initiation of proliferation of quiescent smooth muscle cells, heparin may exert its antiproliferative action by its effect on c-myb. It is an object of the present invention to provide a method for delivery of oligonucleotides to a specific locus in vivo, and thereby to provide localized inhibition of expression of vital genes, oncogenes and genes encoding proteins involved in disease or other pathologic conditions.